U.S. patent number 5,399,494 [Application Number 07/936,829] was granted by the patent office on 1995-03-21 for vibrio cholerae strain cvd103hg.sup.r, method of making same, and vaccines derived therefrom.
This patent grant is currently assigned to The University of Maryland System. Invention is credited to James B. Kaper, Myron M. Levine.
United States Patent |
5,399,494 |
Kaper , et al. |
* March 21, 1995 |
Vibrio cholerae strain CVD103Hg.sup.r, method of making same, and
vaccines derived therefrom
Abstract
Method of isolating deletion mutants of Vibrio cholerae, wherein
the deletion is predetermined by digestion with restriction
endonucleases of known specificity. The deletions are inserted into
the Vibrio cholerae chromosome by in vivo recombination between a
plasmid carrying the desired deletion, with adjacent flanking
sequences, and the Vibrio cholerae chromosome. The invention
includes the isolation and characterization of a new Vibrio
cholerae strain, (ATCC No. 55456), having a deletion in the tox
gene, as defined by Acc I, Xba I, Cla I and/or restriction
endonuclease sites, and carrying a mercury resistance gene. The
invention also includes vaccines for protecting against the
symptoms of cholera as well as methods for achieving this
protection.
Inventors: |
Kaper; James B. (Columbia,
MD), Levine; Myron M. (Columbia, MD) |
Assignee: |
The University of Maryland
System (Baltimore, MD)
|
[*] Notice: |
The portion of the term of this patent
subsequent to June 19, 2007 has been disclaimed. |
Family
ID: |
27413201 |
Appl.
No.: |
07/936,829 |
Filed: |
August 28, 1992 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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757930 |
Sep 12, 1991 |
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581406 |
Feb 17, 1984 |
5135862 |
Aug 4, 1992 |
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472276 |
Mar 4, 1983 |
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Current U.S.
Class: |
435/6.16;
435/909; 536/23.7; 435/477; 435/480; 435/488; 435/482;
435/317.1 |
Current CPC
Class: |
C07K
14/28 (20130101); A61K 39/107 (20130101); C12N
15/01 (20130101); C12N 1/205 (20210501); C12R
2001/63 (20210501); A61K 2039/522 (20130101); Y10S
435/909 (20130101); Y02A 50/30 (20180101) |
Current International
Class: |
C07K
14/195 (20060101); C07K 14/28 (20060101); C12N
15/01 (20060101); C12N 001/21 (); C12N 015/03 ();
C12N 015/31 (); C12R 001/63 () |
Field of
Search: |
;435/172.1,172.3,252.3,320.1,909,252.3,317.1 ;424/93A,92,93.48
;536/23.7 ;935/29,52,56,72,84 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0018154 |
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Oct 1980 |
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EP |
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0095452 |
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Nov 1983 |
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EP |
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0119031 |
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Sep 1984 |
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EP |
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2436818 |
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Aug 1980 |
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FR |
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2032955 |
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May 1980 |
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GB |
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|
Primary Examiner: Furman; Keith C.
Assistant Examiner: Jacobson; Dian C.
Attorney, Agent or Firm: Kile; Bradford E.
Parent Case Text
CROSS REFERENCE TO THE RELATED APPLICATIONS
This application is a continuation-in-part of applicants' U.S.
patent application Ser. No. 07/757,930, filed Sep. 12, 1991, now
abandoned, which is a continuation of Ser. No. 06/581,406, filed
Feb. 17, 1984, issued as U.S. Pat. No. 5,135,862 on Aug. 4, 1992,
which is a continuation-in-part of applicants' U.S. patent
application Ser. No. 06/472,276, filed Mar. 4, 1983, abandoned, the
entire specifications of which are incorporated herein by
reference. The research outlined in this application was supported
by the National Institute of Health.
Claims
What is claimed is:
1. Vibrio cholerae CVD103Hg.sup.r (ATCC No. 55456).
2. A culture of Vibrio cholerae comprising Vibrio cholerae
CVD103Hg.sup.r (ATCC No. 55456).
3. A method of isolating deletion mutants of Vibrio cholerae Inaba
comprising the steps of:
(a) constructing a first plasmid comprising Vibrio cholerae
flanking sequences of the coding sequence for the A.sub.1 subunit
of Vibrio cholerae toxin and a gene for a first selectable marker
of foreign origin ligated to said flanking sequences or coding
sequence, wherein said flanking sequences are of sufficient length
to promote detectable in vivo recombination;
(b) mating virulent strain 569B of Vibrio cholerae with a first
microorganism carrying the first plasmid;
(c) selecting for Vibrio cholerae expressing the first selectable
marker;
(d) mating the selected product of step (c) with a second
microorganism carrying a second plasmid with a second selectable
marker, said second plasmid being incompatible with the first
plasmid in said second microorganism;
(e) selecting for Vibrio cholerae expressing both the first
selectable marker and the second selectable marker to isolate an in
vivo recombinant Vibrio cholerae organism;
(f) mating said in vivo recombinant Vibrio cholerae with a third
microorganism carrying a third plasmid, said third plasmid
comprising flanking sequences of the coding sequence for the
A.sub.1 subunit of Vibrio cholerae toxin, said coding sequence
comprising a deleted fragment of sequence;
(g) selecting for the loss of the first selectable marker;
(h) mating the selected product of step (g) with a fourth
microorganism carrying a fourth plasmid, said fourth plasmid
encoding a gene conferring a resistance to mercury; and,
(i) selecting for Vibrio cholerae expressing the gene conferring
resistance to mercury.
4. The method according to claim 3 wherein said first, second,
third and fourth microorganisms are E. coli strains.
5. The method according to claim 3 wherein said first and second
selectable markers are antibiotic resistance genes.
6. The method according to claim 3 wherein said gene conferring
resistance to mercury in step (i) is incorporated into the
hemolysin gene of 569B.
7. The method according to claim 6 wherein said Vibrio cholerae in
step (i) is CVD103Hg.sup.r (ATCC No. 55456).
8. A Vibrio cholerae having substantially all of the identifying
characteristics of CVD103Hg.sup.r (ATCC No. 55456).
Description
BACKGROUND OF THE INVENTION
Vibrio Cholerae (V.cholerae) is a non-invasive enteropathogen of
the small bowel that does not penetrate the mucosal surface. Local
SIgA mediated immunity at the mucosal surface is therefore
implicated as a protective mechanism. Pathogenic V. cholerae 01
elaborate a protein enterotoxin (also known as cholera enterotoxin,
or choleragen, or cholera toxin) which is responsible for induction
of copious secretion by the intestine resulting in watery diarrhea,
the clinical consequence of cholera infection. Cholera diarrhea can
be extraordinarily severe and result in loss of so much body water
and salts that dehydration, acidosis, shock, and death ensue
without prompt therapy.
The cholera vaccines that have been developed can be broadly
divided into two categories; those aiming to stimulate antitoxic
immunity and those intending to induce antibacterial immunity.
Experiments with animal models support a protective role for either
or both antitoxic and antibacterial immunity. It has been suggested
that when both types of immunity work in unison, there is a
synergistic effect. [Holmgren, J. et al. J. Infect. Dis. 136
Suppl., S105-S1122 (1977); Peterson, J. W. Infect. Immun. 26, 594
(1970); Resnick, I. G. et al. Infect. Immun. 13, 375 (1980);
Svennerholm, A. -M. et al. Infect. Immun. 13, 735 (1976). However,
it appears that protective immunity in humans can be conferred
without such synergistic effect, that is by either antitoxic
immunity or antibacterial immunity [Eubanks, E. R. et al. Infect.
Immun. 15, 533 (1977); Fujita, K. et al. J. Infect. Dis. 125, 647
(1972); Holmgren, J., J. Infect. Dis., supra; Lange, S. et al. Acta
Path. Microbiol. Scand Sect. C 86, 145 (1978); Peterson, J. W.,
supra (1979); Pierce, N. F. et al. Infect Immun. 37, 687 (1982);
Pierce, N. F. et al. Infect. Immun. 21, 185 (1978); Pierce, N. F.
et al. J. Infect. Dis. 135, 888 (1977); Resnick, I. G. et al.,
supra; Svennerholm, A. -M. et al, supra].
KILLED WHOLE CELL VACCINES
1. Parenteral Whole Cell Vaccines
For almost a century, killed whole V. cholerae have been employed
as parenteral vaccines; these vaccines are still commercially
available. Experience with the parenteral whole cell vaccines has
been reviewed recently in Joo, I., "Cholera Vaccines," in Cholera,
(Barua D. and Burrows W., eds.), Saunders, Philadelphia, pp.
333-355 (1974) and in Feeley, J. D. et al., in Cholera and Related
Diarrheas, 43rd Nobel Symp., Stockholm 1978, (O. Oucherlong, J.
Holmgren, eds.) Karger, Basel, pp. 204-210 (1980). Such vaccines
stimulate high titers of serum vibrioicidal antibodies. They also
stimulate increases in intestinal SIgA antibody to V. cholerae
somatic O antigen when given to Pakistanis but not to Swedes
[Svennerholm, A. -M. et al. Infect. Immun. 30, 427 (1980);
Svennerholm, A. -M. et al. Scan. J. Immun. 6, 1345 (1977)]. It has
been suggested that the Pakistani vaccine recipients respond in
this way because they are already immunologically primed from prior
antigenic contact, while persons living in a non-endemic area
(e.g., Sweden) are not. In field trials parenteral killed whole
cell vaccines have been shown to confer significant protection
against the homologous V. cholerae serotype, but usually for a
period of less than one year [Joo, I. supra; Feeley, J. C. , supra;
Svennerholm, A. -M. et al. supra, (1980); Svennerholm, A. -M. et
al. supra, (1977); Mosley, W. H. et al. Bull. Wld. Hlth. Org. 49,
13 (1973); Phillipines Cholera Committee, Bull. Wld. Hlth. Org. 49,
381 (1973)]. There is some evidence to suggest that parenteral
whole cell Inaba vaccine provides good, short term protection
against Ogawa, as well as against Inaba cholera, while Ogawa
vaccine is effective only against Ogawa.
By use of adjuvants, it has been possible to maintain a vaccine
efficacy of approximately 70% for up to one-and-one-half years with
parenteral vaccine (see, e.g., Saroso, J. S. et al. Bull. Wld.
Hlth. Org. 56, 619 (1978)). However, the adverse reactions
encountered at the site of inoculation with adjuvanted vaccines
(which include sterile abscesses) are sufficiently frequent and
severe to preclude routine use of such adjuvanted vaccines.
2. Oral Whole Cell Vaccines
Killed whole vibrios administered orally stimulate the appearance
of local intestinal antivibrio antibody. [Freter, R. J. Infect Dis.
111, 37 (1972); Freter R. et al. J. immunol. 91 724 (1963);
Ganguly, R. et al. Bull. Wld. Hlth. Org. 52, 323 (1975)]. Other
investigators have shown substantial vaccine efficacy, but a large
proportion of the vaccinees developed diarrhea after subsequent
challenge with pathogenic vibrios [Cash, R. A. et al. J. Infect.
Dis. 130, 325 (1974)].
TOXOIDS
Immunizing agents intended to prevent cholera by means of
stimulating antitoxic immunity include:
1) Formaldehyde-treated cholera toxoid
2) Glutaraldehyde-treated cholera toxoid;
3) Purified B subunit; and
4) Procholeragenoid (with or without formaldehyde treatment).
1. Formaldehyde-Treated Cholera Toxoid
Treatment of purified cholera toxin in vitro with formaldehyde
eradicates its toxicity, resulting in a toxoid that exhibits little
toxic biological activity but stimulates antitoxic antibodies
following parenteral immunization of animals. However, when the
first toxoid of this type was administered to monkeys and man as a
parenteral vaccine, the toxoid reverted to partial toxicity causing
unacceptable local adverse reactions at the site of inoculation
[Northrup, R. S. et al. J. Infect. Dis. 125, 471 (1972)]. An
aluminum-adjuvanted formalinized cholera toxoid has been
administered parenterally to Bangladeshi volunteers, including
lactating mothers, but no field trials with this vaccine have been
undertaken [Merson, M. H. et al. Lancet I, 931 (1980)].
Formalinized cholera toxoid prepared in the presence of glycine has
also been tried by the parenteral route, but the vaccine showed no
evidence of efficacy [Ohtomo, N. In Proceedings of the 12th Joint
Conference on Cholera, U.S.-Japan Cooperative Medical Science
Program, Sapporo (Fukumi H., Zinnaka Y., eds.) pp. 286-296 (1976);
Noriki, H. In Proceedings of the 12th Joint Conference on Cholera ,
U.S.-Japan Cooperative Medical Science Program, Sapporo (Fukumi H.,
Zinnaka Y., eds.) pp. 302-310 (1976)].
2. Glutaraldehyde-Treated Cholera Toxoid
Methods have been developed for the large-scale preparation of a
glutaraldehyde-treated cholera toxoid that is essentially free of
contaminating somatic antigen [Rappaport, E. S. et al. Infect.
Immun. 14, 687 (1976)]. It was hoped that this antigen could be
used to assess in a "pure" manner the protective role of antitoxic
immunity alone. A large-scale field trial of this toxoid given as a
parenteral vaccine was carried out in Bangladesh in 1974 [Curlin,
G. et al. In Proceeding of the 11th Joint Conference on Cholera,
U.S.-Japan Cooperative Medical Science Program. pp. 314-329, New
Orleans, (1975)]. The toxoid stimulated high titers of circulating
antitoxins in Bangladeshi recipients. Two waves of cholera, El Tor
Inaba followed by El Tor Ogawa, struck the field area allowing a
fair evaluation of vaccine efficacy. A protective effect could be
demonstrated in only one age group and was restricted to the period
of the Inaba epidemic, so that glutaraldehyde-treated cholera
toxoid given alone as a parenteral vaccine provided little
protection and was substantially inferior to similar field trials
in the same population with parenteral killed whole cell
vaccines.
The use of glutaraldehyde-treated cholera toxoid as an oral vaccine
has been investigated on the assumption that toxoid given by this
route might be more efficient by stimulating intestinal antitoxin
[Levine, M. M. et al. Trans. Roy. Soc. Trop. Med. Hyg. 73, 3,
(1979)]. Two groups of volunteers were immunized with three 2.0
mg., or three 8.0 mg doses of toxoid given directly into the small
intestinal lumen (via intestinal tube) at monthly intervals. The
vaccinees and unimmunized controls then participated in
experimental cholera challenge studies. In neither challenge study
was the attack rate or severity of diarrhea significantly
diminished in the vaccinees when compared with controls. The lack
of efficacy of oral glutaraldehyde-treated cholera toxoid may be
due to the fact that the capacity of B subunits to bind to GM1
ganglioside is greatly diminished as a consequence of toxoiding
with glutaraldehyde.
3. Purified B Subunit
Cholera enterotoxin is composed of two subunits designated A and B.
The A subunit induces the enzymatic changes which lead to fluid
secretion, while the non-toxic B subunit is the immunogenic moiety
that binds to the receptor for toxin (GM1 ganglioside) on
intestinal epithelial cells [Holmgren, J. Nature 292, 413 (1981)].
It has been shown that purified B subunit given either orally or
parenterally to Bangladeshis stimulates the appearance of SIgA
antitoxin in intestinal fluid, a result attributable to
immunological priming in a cholera-endemic area [Svennerholm, A.
-M. et al. Lancet I, 305 (1982)].
The major advantages of B subunit oral vaccine to stimulate
antitoxic immunity include its complete safety (there is not
potential for reversion to toxin as exists with toxoids) and
retention of its capacity to adhere to toxin receptors on
enterocytes. Animal studies suggest that it is less potent than
native holotoxin in stimulating antitoxin [Pierce, N. F. supra,
(1982)].
It will be understood that the purified B subunit can be used, if
at all, in conjunction with e.g. oral killed vibrios as a
combination oral vaccine intended to stimulate both antibacterial
and antitoxic antibodies.
4. Procholeragenoid
Procholeragenoid is the large molecular weight toxoid (ca.
1,000,000 MW) that results when cholera enterotoxin is heated at
65.degree. C. for at least five minutes [Finkelstein, R. A. et al.
J. Immunol. 107, 1043 (1971)]. It is immunogenic while retaining
less that 5% of the biological toxic activity of the parent toxin.
Heating for longer times (e.g., 25 minutes) produces less
biological toxicity [Germanier, R. et al. Infect. Immul 13, 1692
(1976)], and subsequent treatment with formaldehyde completely
abolishes residual biological toxicity. The resultant
formaldehyde-treated procholeragenoid is at least as potent as the
parent toxin in stimulating serum antitoxin following immunization
of rabbits. Swiss volunteers developed brisk serum antitoxin
responses following parenteral immunization with 10, 30, or 100 mcg
doses of formaldehyde-treated procholeragenoid [Germanier, R. et
al. J. Infect. Dis. 135. 512 (1977)]. No notable adverse reactions
were observed.
As an oral antigen procholeragenoid is more immunogenic when given
in the form without formaldehyde-treatment. In dogs, untreated
procholeragenoid is tolerated well as an oral vaccine; oral doses
(with NaHCO.sub.3) up to 500 mcg do not cause diarrhea. Five 500
mcg doses spaced over 42 days stimulate significant protection in
dogs against oral challenge with pathogenic V. cholerae. Doses of
50 mcg and 200 mcg with NaHCO.sub.3 have been given to groups of
six and four adult volunteers, respectively, without eliciting
adverse reactions.
It will be understood that procholeragenoid can be used in
conjunction with e.g. killed vibrios or other relevant antigens
capable of stimulating antibacterial immunity so that the antitoxic
immunity induced by procholeragenoid is enhanced.
COMBINATION VACCINES
The major attraction of non-living, oral cholera vaccine is its
safety. An oral vaccine consisting of a combination of antigens,
intending to stimulate both antibacterial and antitoxic immunity,
would be most likely to succeed for the following reasons: Toxoid
vaccines that stimulate purely antitoxic immunity have not been
shown to be efficacious in protecting man against cholera, although
they may protect animal models. In addition, oral or parenteral
killed whole cell vaccines that stimulate no antitoxic immunity
provide significant protection against cholera in man, albeit for a
short period of time. Furthermore, combinations of antigens (such
as crude cholera toxin, or toxin plus lipopolysaccaride) that
stimulate both antitoxic and antibacterial immunity, give
synergistic protection.
Two studies so far have been carried out in many with combination
vaccines. In the first, nine volunteers who ingested
glutaraldehyde-treated cholera toxoid (2 mg weekly for four weeks)
plus killed El Tor Inaba vibrios (10.sup.10 vibrios twice weekly
for four weeks) were challenged after one month with 10.sup.6
pathogenic El Tor Inaba vibrios, along with six unimmunized
controls. Diarrhea occurred in only two of nine vaccinees, versus
four of six controls (vaccine efficacy 67%) and illness was clearly
attenuated in the two ill vaccinees. More pertinent, perhaps, is
the observation that V. cholerae could be directly cultured from
stools of only two of nine vaccinees, versus six of six controls.
This demonstrates that immunologic mechanisms impeded the
proliferation of vibrios.
More recently, three doses of B subunit/killed whole cell vaccine
was given to adult volunteers who participated in a vaccine
efficacy challenge. The combination vaccine was give on days 0, 14,
and 28. Each of the three doses of vaccine contained 0.5 mg of
purified B subunit and 2.times.10.sup.11 killed V. cholerae
(5.times.10.sup.10 classical Inaba, 5.times.10.sup.10 classical
Ogawa, and 1.times.10.sup.11 El Tor Inaba).
A group of eleven volunteers immunized with this combination
vaccine were challenged one month after their last dose with
10.sup.6 pathogenic V. cholerae El Tor Inaba, along with seven
control volunteers. Diarrhea occurred in seven of seven controls,
but in only four of eleven vaccinees (p=0.01). The illness in the
four vaccinees was definitely milder.
Thus, results of studies with oral toxoid/killed whole cell vaccine
combinations demonstrate a measurable degree of efficacy. The
protective vaccine efficacy, however, is only moderate (55-65%) and
multiple doses are required to induce the protection.
ATTENUATED V. CHOLERAE VACCINES
Both classical and El Tor clinical cholera infections stimulate a
high degree of protective immunity for at least three years in
North American volunteers [Cash, R. A. et al., supra (1974);
Levine, M. M. et al., supra (1979); Levine, M. M. et al.
"Volunteers studies in development of vaccines against cholera and
enterotoxigenic Escherichia coli: a review," in Acute Enteric
Infections in Children: New Prospects for Treatment and Prevention.
(T. Holm, J. Holmgren, M. Merson, and R. Mollby, eds.) Elsevier,
Amsterdam, pp. 443-459 (1981); and Levine, M. M. et al. J. Infect.
Dis. 143, 818 (1981)]. Based on these observations in volunteers,
perhaps the most promising approach toward immunologic control of
cholera may be with attenuated non-toxigenic V. cholerae strains
employed as oral vaccines.
1. Naturally-Occurring Strains
Non-toxigenic V. cholerae 01 strains isolated from environmental
sources in India and Brazil have been evaluated in volunteers as
potential vaccine candidates with disappointing results. They
either failed to colonize the intestine of man, or did so
minimally; vibrocidal antibody responses were meager, and they
failed to provide protection in experimental challenge studies
[Cash, R. A. et al. Infect. Immun. 10, 762 (1974); Levine M. M. et
al. J. Infect. Dis. 145, 296 (1982)]. Many of these strains appear
to lack the toxin gene, as measured by hybridization with a
radioactive DNA probe [Kaper, J. B. et al. Infect. Immun. 32, 661
(1981)].
2. Mutagenized Attenuated Strains
Classical Inaba 569B has been mutagenized with nitro-soguanide
(NTG) and a hypotoxinogenic mutant isolated [Finkelstien, R. A. et
al. J. Infect. Dis. 129, 117 (1974); Holmes, R. K. et al. J. Clin.
Invest. 55, 551 (1975). This mutant strain, M13, was fed to
volunteers. Diarrhea did not occur but the strain colonized poorly.
Challenge studies demonstrated that some protective efficacy was
conferred by immunization with multiple doses [Woodward, E. et al.
Develop. Biol. Stand. 33., 108, (1976)].
El Tor Ogawa 3083 has also been mutagenized [Honda, T. et al. Proc.
Nat. Acad. Sci. 76, 2052 (1979)]. Brute force selection and
analysis of thousands of colonies yielded one isolate that
continued to produce the immunogenic B subunit while failing to
produce detectable A subunit or holotoxin. The one isolate, Texas
Star-SR, fulfilled these criteria. Texas Star-SR produces normal or
increased amount of B subunit but is negative in assays for
holotoxin activity or A subunit activity.
Texas Star-SR has been extensively evaluated in volunteers (see,
e.g., Levine M. M. et al. Acute Enteric, supra. (1981)). Groups of
five volunteers received two 10.sup.9 organism doses one week apart
and eighteen more volunteers ingested two 2.times.10.sup.10
organism doses one week apart. Some degree of diarrhea was seen in
sixteen of the sixty-eight vaccinees (24%). In only one individual
did the total stool volume exceed 1.0 liter (1464 ml). Typically,
the vaccine-induced diarrhea consisted of two or three small, loose
stools totalling less than 400 ml in volume. Vaccine organisms were
recovered from coprocultures of approximately one-half of the
vaccine recipients. Where jejunal fluid was cultured (recipients of
doses of 10.sup.8 or more vaccine organisms), cultures were
positive in thirty-five of forty-six vaccinees (76%). Hundreds of
Texas Star clones recovered from copro-cultures and jejunal fluid
cultures were examined for cholera holotoxin by the sensitive Y-1
adrenal cell assay; none were positive.
Significant rises in serum antitoxin were detected in only 29% of
the vaccinees; however, 93% manifested significant rises in serum
vibriocidal antibody and the titers were substantially close to
those encountered following infection with pathogenic V. cholerae.
In experimental challenge studies in volunteers, Texas Star-SR was
found to confer significant protection against challenge with both
EL Tor Ogawa And El Tor Inaba vibrios. One or two doses of Texas
Star-SR attenuated oral vaccine confers good protection against El
Tor cholera.
It is clear that the use of attenuated strains has intrinsic
advantages since such strains mimic infection-derived immunity to
cholera. However, the Texas Star-SR strains suffers from certain
drawbacks. To begin with, mutagenesis (e.g., with nitrosoguanidine)
induces multiple mutations, not all of which are necessarily
recognized. Furthermore, the precise genetic lesion that is
presumed to be responsible for the attenuation of Texas Star-SR is
not known. In addition, Texas Star-SR may revert to virulence, like
any pathogen mutated with nitrosoguanidine.
Applicants of the present invention have isolated, by a novel
method, deletion mutants of a virulent strain of Vibrio cholerae
known to produce both immunity and disease in volunteers. The
deletions are restriction endonuclease fragments. The vaccine
strains of the present invention have been specifically altered
through the use of recombinant DNA techniques to render the
avirulent without affecting other components necessary for
immunity. This attenuation was accomplished by using restriction
endonucleases which cleave the DNA of the bacterium at specific
sites, to specifically delete the genes responsible for cholera
toxin (i.e., the ctx gene). Plasmids carrying the ctx gene were
digested with restriction endonucleases to delete the ctx gene, but
were constructed to retain extensive lengths of flanking DNA of the
V. cholerae chromosome. Conjugal gene transfer of the plasmids into
V. cholerae yielded an avirulent V. cholerae strain carrying the
extrachromosomal copies of the plasmids. Subsequent conjugation
with cells having other plasmids produced, after appropriate
selection of selectable plasmid markers, V. cholerae strains having
deletions in the ctx regions. Such nontoxigenic deletion mutants
would then be capable of colonizing the small intestine and
stimulating local, protective immunity directed against the
bacterial cell. After the transient colonization episode, the
vaccine would be protective against subsequent disease symptoms
which would normally occur upon challenge with virulent toxigenic
V. cholerae strains.
The genes for V. cholerae toxin have been cloned [Pearson, G. D. N.
et al. Prod. Nat. Acad. Sci. 79, 2976 (1982); Kaper, J. B. et al.
Amer. Soc. Micribiol. Abstr. Annu. Meeting, Atlanta, Georgia, 36
(1982); Kaper, J. B. et al. Symposium on Enteric Infections in Man
and in Animals: Standardization of Immunological Procedures,
Dublin, Ireland, Abstract No. 2.5 (1982)]. Toxin structural gene
deletion mutants of V. cholerae have been isolated, but only by
infection with mutagenic vibriophages capable of integration at
random sites along to chromosome [Mekalanos, J. J. et al. Proc.
Nat. Acad, Sci. 79, 151, (1982)]. Recombination in Vibrio cholerae
has been reported, but it has not been used to isolate restriction
fragment deletions in the ctx genes for vaccination purposes
[Parker, C. et al. J. Bact. 112, 707 (1972); Johnson, S. R. et al.
Molec. Gen. Genet. 170, 93 (1979); Sublett, R. D. et al. Infect.
Immun. 32 1132 (1981) and Thomson, J. A. et al. J. Bact. 148, 374
(1981)].
BRIEF DESCRIPTION OF THE INVENTION
A culture of Vibrio cholerae is described comprising a Vibrio
cholerae strain having a restriction endonuclease fragment of DNA
deleted to confer avirulence and to retain capacity to colonize the
intestine of a host animal. One isolated deletion mutant
encompasses a deletion in the tox gene, as defined by Acc I
restriction endonuclease sites.
A method of isolating such deletion mutants of Vibrio cholerae is
also described, comprising the steps of
(a) constructing a first plasmid comprising Vibrio cholerae
flanking sequences of one or more deleted restriction endonuclease
fragments and a gene for a first selectable marker of foreign
origin ligated to said flanking sequences to substitute for and to
be in the place of said deleted fragment, wherein said sequences
are of sufficient length to promote detectable in vivo
recombination;
(b) mating a virulent strain of Vibrio cholerae with a first
microorganism carrying the first plasmid;
(c) selecting for Vibrio cholerae expressing the first selectable
marker;
(d) mating the selected product of step (c) with a second
microorganism carrying a second plasmid with a second selectable
marker, said second plasmid being incompatible with the first
plasmid; and
(e) selecting for Vibrio cholerae expressing both the first
selectable marker and the second selectable marker.
The Vibrio cholerae deletion mutants of this invention are useful
in vaccination against cholera.
One Vibrio cholerae strain of the present invention, designated
CVD103Hg.sup.r, confers substantially close to 100% efficacy in
humans against subsequent disease symptoms after challenge with a
strain of a similar serotype.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. V. cholerae N16961 (pJBK55) (Ap.sup.r)
FIG. 2. Processes of crossing-over and conjugal gene transfer to
construct V. cholerae JBK56.
FIG. 3. V. cholerae JBK56.
FIG. 4. Scheme for construction of JBK21.
FIG. 5. Scheme for construction of pJBK54.
FIG. 6. Scheme for construction of V. cholerae JBK56.
FIG. 7. Recombination in vivo by cross over and elimination of tox
gene.
FIG. 8. Scheme for construction of pJBK51.
FIG. 9. Scheme for construction of pCVD14 and pCVD15.
FIG. 10. Scheme for construction of pJBK108.
FIG. 11. Scheme for construction of pJBK107.
FIG. 12. DNA sequence of (top) the Xba I and Cla I sites, which
determine the ends of the deleted Xba I-Cla I 550 bp fragment of
the A subunit in Ogawa 395, and for (bottom) the junction in CVD101
after deletion of this fragment and insertion of an Xba I
linker.
Abbreviations for restriction endonuclease sites in the drawings
are as follows:
A=Acc I restriction endonuclease site
B=Bgl II restriction endonuclease site
C=Cla I restriction endonuclease site
E=Eco RI restriction endonuclease site
H=Hind III restriction endonuclease site
P=Pst I restriction endonuclease site
S=Sal I restriction endonuclease site
X=Xba I restriction endonuclease site
Other abbreviations in the drawings and elsewhere include:
Ap=Ampicillin resistance gene
Ap.sup.r =Ampicillin resistance phenotype
Ap.sup.s =Ampicillin sensitive phenotype
Chrom=Chromosome
Cm=Chloramphenicol resistance gene
CT=Cholera toxin
CTA=gene for A subunit of cholera toxin
CTB=gene for B subunit of cholera toxin
kb=Kilobases
p=plasmid
Su=Sulfonamide
Su.sup.r =Sulfonamide resistance phenotype
Tc=tetracycline
Tc.sup.s =tetracycline sensitive phenotype
Tp=Trimethoprin
DETAILED DESCRIPTION OF THE INVENTION
The principle of the present invention is the isolation of a Vibrio
cholerae vaccine strain specifically altered through recombinant
DNA technology to render it avirulent without affecting other
components necessary for immunity. This attenuation was
accomplished by restriction endonuclease digestion of plasmids
carrying appropriate V. cholera sequences, to specifically delete
the genes coding for cholera toxin, or portion thereof. Conjugal
gene transfer of these digested plasmids, followed by procedures
for selecting in vivo recombinants with virulent host V. cholera,
resulted in strains without the toxin genes or portion thereof. It
will be understood that the methods of the present invention are
applicable to the isolation of other deletion mutants of virulent
V. cholerae, or to the isolation of strains having all or part of
such deleted sequences reintroduced into the V. cholerae cell.
The starting material for the vaccine was the toxigenic Vibrio
cholerae strain N16961, which has been demonstrated to produce in
volunteers both typical diarrheal disease and strong, protective
immunity to subsequent infection [Levine, M. M. et al., Acute
enteric, supra. 1981]. The region of the bacterial chromosome which
was found to be responsible for production of cholera toxin was
cloned into the plasmid cloning vehicle pBR325, after screening
Hind III digest of V. cholerae with an E. coli heat-labile
enterotoxin gene probe [Kaper et al. Amer. Soc., supra; Kaper et
al. Symposium, supra]. The V. cholerae chromosome fragment was
found to contain all genes necessary for toxin production. Next,
this chromosomal region was then analyzed and mapped for the exact
portions containing the toxin genes [Kaper, J. B. et al. Lancet II,
1162 (1981)]. Restriction enzymes were employed to cut out the DNA
fragments containing these genes and a DNA fragment encoding a
selectable marker (e.g., resistance to ampicillin) was inserted by
ligation. The ampicillin resistance gene and the flanking Vibrio
DNA were then cloned in a derivative of pRK290 which can be
transferred from E. coli to V. cholerae. The resulting plasmid,
pJBK55, was transferred from E. coli K-12 to V. cholerae N16961 by
conjugation.
The resulting strain, V. cholerae N16961 (pJBK55) (Ap.sup.r)
contained a region in its chromosome having intact toxin genes and,
in an extrachromosomal state, a plasmid containing this same region
with the toxin genes deleted and a gene for ampicillin substituted.
(See FIG. 1.) At a low frequency, perhaps one in 10.sup.6 to one in
10.sup.8, the identical regions flanking the chromosomal toxin
genes and the extrachromosomal (plasmid) ampicillin resistance gene
will exchange and "cross over" or undergo in vivo recombination so
that the region of DNA containing the resistance gene displaces the
toxin gene on the chromosome (FIG. 2). This rare event is selected
by testing a mixture of mutated and nonmutated cells for individual
cells which are able to serve as host for an incoming incompatible
plasmid [Ruvkun, G. B. et al. Nature 289, 85 (1981)]. Plasmids are
divided into groups designated A through W, the members of which
cannot stably coexist with each other. For example, a plasmid of
incompatibility group P cannot be stably maintained in the same
cell as another P group (Inc P) plasmid. Thus, Inc P plasmids, such
as R702, which confers resistance to sulfonamide, cannot be
maintained in a cell which has another Inc P plasmid such as PRK
290, pJBK45, or pJBK55. Therefore, R702 can be maintained in a
strain in which the ampicillin resistance has recombined into the
chromosome but not one in which an Inc P plasmid (e.g. pJBK55) is
replicating extrachromosomally. By mating an E. coli strain
containing Inc P R702 (sulfonamide resistance) and V. cholerae
pJBK55 (ampicillin resistance) and selecting for V. cholerae which
are resistant to both ampicillin and sulfonamide, colonies are
isolated in which the sulfonamide resistance is mediated
extrachromosomally by p702 and the ampicillin resistance is
mediated chromosomally through substitution of the ampicillin
resistance gene for the toxin gene (FIG. 3). One such strain,
designated V. cholerae JBK56 was isolated and when tested for toxin
production was found to be nontoxinogenic.
The final version of the vaccine strain, JBK70, was produced by
substituting resistance to ampicillin, a therapeutically useful
antibiotic, with resistance to mercury. This substitution was
accomplished by cloning a gene for mercury resistance directly into
the ampicillin resistant gene of pJBK55, thereby inactivating
ampicillin resistance and conferring mercury resistance. The
resulting plasmid, pJBK66 was also incompatible with R702 and was
transferred to V. cholerae JBK56. A mutant in which the mercury
resistance was recombined into the chromosome was selected using
the Inc P plasmid R702 and selecting for V. cholerae which were
ampicillin sensitive, mercury resistant, and sulfonamide resistant.
A spontaneous derivative was later selected which was cured of
pR702. The final mutant, JBK70, was nontoxinogenic and resistant to
mercury only.
The vaccine strain V. cholerae JBK70 is one of the Inaba serotype.
The other major serotype of V. cholerae is the Ogawa serotype. It
is expected that a vaccine prepared from one serotype will protect
against the other serotype. In the event that this is not the case,
a live vaccine strain can be prepared from an Ogawa serotype and
confer protection in volunteers [Levine, M. M. et al. Acute
enteric, supra (1981)]. The exact mutation created in strain V.
cholerae Inaba JBK56 was recreated in strain E7946 by directly
transferring the region of the chromosome containing the ampicillin
resistance in place of the toxin gene in JBK56 into E7946 through
genetic recombination mediated by P, the sex factor of V. cholerae
[Parker, C. et al., supra]. The P factor, which is distinct from
Inc P plasmid, was transferred into JBK56 and was then mated with a
rifampin resistant mutant of E7946. By selection of a mutant which
was resistant to both ampicillin and rifampin, a vaccine strain was
isolated which was of the Ogawa serotype with the toxin genes
completely deleted.
If antibacterial immunity is insufficient for protection, then an
antitoxic component can be added by adding back the genes for
production of cholera toxin B but not A subunit. This has been
accomplished by cloning the B subunit gene into the cloning vector
pMS9. The resulting plasmid, pJBK51, produces high levels of B
subunit and was reintroduced into the nontoxic vaccine strain V.
cholerae JBK70 to make an attenuated vaccine strain JBK70 (pJBK51)
which fails to produce the A subunit.
The vaccine strains of the present invention are derived inter alia
from V. cholerae N16961 having the serotype Inaba. It will be
understood that other strains or other biotypes and serotypes can
be used to substitute for N16961 to produce vaccine strains having
specific deletions in the tox gene or genes, or in other locations
along the V. cholerae chromosome. Since the object of isolating
such vaccine strains is to mimic the infection process without the
associated pathological phenomena, site-directed mutagenesis of
virulent strains, as described in this application, produces
substantial possibilities in the prophylactic vaccination against
cholera.
For example, applicants have produced another V. cholerae vaccine
strain CVD101, characterized by a deletion of most of the A subunit
gene present in 2 copies of the tox gene. It is expected that the
efficacy of CVD101 is substantially close to 100%, since the parent
strain 395 confers 100% efficacy.
Construction of CVD101 followed in general the principles outlined
supra, e.g. the construction of JBK70, except that the resulting
CVD101 had no resistance gene that needed curing. The final step in
isolating an in vivo recombinant included a scheme for selecting
sensitivity to an antibiotic e.g. tetracycline sensitivity, whereas
the parent strain had inserted at the location of the A gene of CT
a tetracycline resistance gene. It will be understood that such
antibiotic sensitivity is another example of a selectable
marker.
Applicants have produced another V. cholerae vaccine strain CVD103,
which is a derivative of V. cholerae strain 569B. The same mutation
was made in 569B to derive CVD103 as was made in CVD101, which is
described above. The procedure used to prepare CVD103 is the same
procedure used to prepare CVD101, except that 569B was used rather
than 395. CVD103 is a derivative of wild-type strain classical
Inaba 569B that has a fragment of DNA coding for the A.sub.1
subunit of cholera toxin deleted.
Applicants have produced another V. cholerae vaccine strain,
CVD103Hg.sup.r, which is a derivative of CVD103. CVD103Hg.sup.r has
a mercury resistance gene incorporated in the chromosome of
CVD103.
Production of vaccine strains can be performed by a variety of
methods, including the following: Vibrio cholerae is subcultured
from stock cultures into brain/heart infusion agar (BHIA) and grown
at 37.degree. C. overnight. Identity is tested with group- and
type-specific antisera and twenty to thirty colonies are suspended
in BHI broth. Preincubated BHIA plates are inoculated with BHI
suspension. After incubation for five to six hours, each plate is
harvested with 5 ml of sterile saline buffered to pH 7.2.+-.0.1.
Harvested organisms are centrifuged in the cold at 750 g for ten
minutes, resuspended and washed twice in four-times the original
volume. The suspension is standardized spectrophotometrically and
diluted to approximately the number of organisms required for
vaccination (ca 10.sup.6, which varies depending on the results of
volunteer studies). Replicate, pour-plate quantitative cultures are
made of the inocula before and after challenge to confirm inoculum
size. The final inoculum is examined with Gram's stain and
agglutinated with homologous antiserum prior to feeding.
The Vibrio cholerae strains of the present invention can be
administered by the oral route. Two grams of NaHCO.sub.3 are
dissolved in five ounces of distilled water. Volunteers drink four
ounces of the NaHCO.sub.3 /water; one minute later the volunteers
ingest the vibrios suspended in the remaining one ounce of
NaHCO.sub.3 /water. Volunteers are NPO ninety minutes pre- and
postinoculation.
With regard to safety, the major concern is that the vaccine strain
does not revert to toxigencity (i.e., produce intact cholera toxin)
which could cause disease. The two major assays for testing toxin
are the Y-1 adrenal cell assay [Sack, D. A. et al. Infect. Immjun.
11, 334 (1975)] and the enzyme-linked immunosorbent assay (ELISA)
[Sack, D. A. et al. J. Clin. Micro. 11, 35 (1980)]. The vaccine
strain (JBK70) has been repeatedly tested in these two assays and
found to be negative each time. Far more important, however, are
the genetic assays performed for the presence of toxin genes. The
DNA for cholera toxin genes can be radioactively labeled and used
as a specific probe to identify other cholera toxin genes in the
strain, according to the method of Southern, E. M. J. Mol. Bio. 98,
503 (1975). When tested by this method, the vaccine strain
described in the invention possesses no detectable genetic material
that can encode cholera toxin. The vaccine has also been tested in
an infant mouse model, according to Baselski, V. et al. Infect.
Immun. 15, 704 (1977). After repeated (ten in all) serial passages,
no fluid accumulation (i.e., evidence of disease, has been found.
As expected, JBK70 was found to colonize the infant mouse
intestine.
In the examples that follow, any of the techniques, reactions, and
separation procedures are already well known in the art. All
enzymes, unless otherwise stated, are available from one or more
commercial sources, such as New England BioLabs--Beverly, Mass.;
Collaborative Research--Waltham, Mass.; Miles
Laboratories--Elkhart, Ind.; Boehringer Biochemicals
Inc.--Indianapolis, Ind.; and Bethesda Research
Laboratory--Rockville, Md., to mention a representative few.
Buffers and reaction conditions for restriction enzyme digestion
are used according to recommendations supplied by the manufacturer
for each enzyme, unless indicated otherwise. Partial digestions
with restriction enzymes are carried out using a reduced enzyme
concentration which must be predetermined from preliminary
experiments for each enzyme batch. Standard methodology for other
enzyme reactions, gel electrophoresis separations, and E. coli
transformation may be found in Methods in Enzymology Volume 68, Ray
Wu, editor, Academic Press (1979). Another standard reference is
Maniatis, T. et al. Molecular Cloning, Cold Spring Harbor (1982).
Bacteria were grown according to procedures generally described in
Miller, Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory (1972) Vibrio cholerae were propagated according to
procedures generally described in Lennett, E. A. et al., eds.,
Manual of Clinical Microbiology 3rd Edition, American Society of
Microbioloy, Washington (1980). E. coli and V. cholerae were mated
according to procedures generally described in Johnson, Steven R.
et al. J. Bact. 137, 531 (1979); and Yokata, T. et al. J. Bact.
109, 440 (1972).
The strains of this invention have been deposited at the American
Type Culture Collection, located in Rockville, Md., prior to
execution of the present application. The strains deposited are V.
cholerae JBK56, V. cholerae JBK70, V. cholerae N16961, V. cholerae
JBK70 (pJBK51), V. cholerae Ogawa 395, CVD101, CVD103Hg.sup.r which
have ATCC accession numbers 39,317, 39,318, 39,315, 39,316, 39,541,
39,540 and 55,456 respectively.
EXAMPLE 1
Construction of a Plasmid Having a Selectable Marker Gene Inserted
to Replace to Toxin Genes
The plasmid JBK16 contains a 4 kb PstI-BglII fragment of the
chromosome containing the toxin genes. The toxin genes are flanked
by Acc I sites and contain an internal Acc I site. JBK16 was
digested to completion with Acc I and the Acc I fragments
containing the toxin genes were separated from the rest of the
plasmid. The remaining overlapping or "sticky" Acc I ends were made
blunt-ended by "filling in" with the Klenow fragment of E. coli
polymerase (i.e., the single-stranded DNA remaining after Acc I
digestion were made double-stranded with flush ends). A gene
encoding ampicillin resistance was purified from the plasmid
pREG153 (pREG153 is a derivative of pREG151 [Weiss, A. et al. J.
Bact. 152, 549-552] altered by substitution of ampicillin
resistance for trimethoprin resistance and addition of .lambda. cos
sequences) and the "sticky" ends "filled in" as above. This
fragment was then ligated to the vibrio DNA so that the Ap
resistance gene was in exactly the same place as the now-deleted
toxin genes, flanked by the same vibrio sequences. The resulting
plasmid was designed pJBK21 (FIG. 4) containing the deletion toxin
region and the Ap resistance gene.
EXAMPLE 2
Addition of Flanking Homogeneous Sequences, Followed by Conjugal
Gene Transfer into V. cholerae
To insure the specific insertion into the chromosome of the
deletion in pJBK21, approximately 7,000 bp of additional DNA was
added to each end of the PstI-BglII fragment from pJBK21. (The
probability of the homologous recombination event occurring
increases with increasing length of flanking homologous sequences.)
To achieve this, an approximately 18 kb fragment was cloned from
the chromosome of N16961. This clone was designated pJBK44 and
contains the 4 kb PstI-BglII tox gene fragment flanked by
approximately 7kb of DNA on each side (see FIG. 5). The plasmid
pJBK21 was partially digested with Pst I so that only one of the
Pst sites would be cut (an additional Pst site was present within
the ampicillin resistance gene) followed by digestion with Bgl II
to isolate the 4 kb PstI-BglII fragment containing the deleted
toxin region and the Ap resistance region. The plasmid pJBK44
containing the ca 18 kb vibrio fragment was partially digested with
Bgl II so that only one of the 4 Bgl II sites present would be cut.
This partial digestion was followed by complete digestion with Pst
I and the resulting fragments separated by electrophoresis through
0.3% agarose. The separated fragments were then purified and
analyzed and one fragment was found which contained all of the
sequences of pJBK44 except for the 4 kb. PstI-BglII tox gene
fragment (see FIG. 5.). This fragment representing the flanking DNA
was then ligated to the Pst-Bgl fragment from pJBK21 containing the
ampicillin resistance gene. The resulting plasmid, pJBK54,
contained approximately 17 kb of Vibrio chromosome with an
ampicillin resistance gene substituted for the deleted toxin
genes.
The modified chromosomal region was the cloned into a plasmid which
can be readily mobilized in V. cholerae. The plasmid pRK290 [Ditta,
G. et al. Proc. Nat. Acad. Sci. 77, 7347 (1980)] belongs to the
plasmid incompatibility group P and possesses a single Eco RI site
into which pJBK54 was cloned (FIG. 6). The resulting plasmid pJBK55
was then mated into V. cholerae N16961 using the conjugative
plasmid pRK2013, yielding V. cholerae N16961 (pJBK55)
(Ap.sup.r).
EXAMPLE 3
Recombination in vivo
The mutant toxin genes, after conjugal gene transfer as described
in Example 2, now existed extrachromosomally in V. cholerae strain
N16961 (see FIG. 1). At a very low frequency (perhaps 10.sup.-6 to
10.sup.-8) the homologous flanking sequences base pair and cross
over into the chromosome (see FIG. 7). This rare event will result
in the substitution of the deleted toxin region on the plasmid for
the ctx genes on the chromosome. To select for this rare event, the
plasmid incompatibility phenomenon was exploited [Ruvkin, G. B.,
supra]. Plasmids can be divided into incompatibility groups,
designated A through W, on the basis of their ability to be stably
maintained together in the same cell. If two plasmids cannot be
stably maintained together in the same cell, they are incompatible
and belong to the same incompatibility group presumably because
they utilize the same replication mechanism in the cell. By
selectively using an antibiotic resistance present on one plasmid
but not on the other, it is possible to select which of two
incompatible plasmids will be maintained. The plasmid pJBK55,
because of its pRK290 origin, belongs to the (Inc) group P. The
plasmid R702 also belongs to the Inc P group and encodes resistance
to kanamycin, tetracycline, sulfonamide, and streptomycin, but not
ampicillin. By mating pR702 (Su.sup.R) into N16961 (pJBK55)
(Ap.sup.R) and selecting on media containing both ampicillin and
sulfonamide, selection was made for cells in which the ampicillin
resistance gene had been incorporated into the chromosome and the
sulfonamide resistance marker remains on the plasmid R702, since
pR702 and pJBK55 are incompatible (see FIG. 2). The resultant
strain JBK56 (FIG. 3) was ampicillin resistant, and toxin negative
when tested in Y-1 adrenal cells and by Gm.sub.1 ELISA.
Furthermore, when chromosomal DNA was hybridized to DNA probes
containing cloned cholera toxin (CT) genes, JBK56 was negative,
suggesting that the toxin genes were completely deleted.
The antibiotic resistance encoded on R702 was eliminated by
selecting a spontaneously cured derivative lacking the plasmid
(this occurred at a frequency of about 1 in 2,000).
EXAMPLE 4
Elimination of the Selectable Marker of Example 1
To eliminate the ampicillin resistance, a derivative of pJBK55 was
constructed in which genes encoding resistance to mercury (Hg) from
R100 were cloned into the Pst site of the Ap gene, thereby
insertionally inactivating the ampicillin resistance gene. This
derivative was then mated into V. cholerae JBK56, followed by pR702
and selection made as above for Hg.sup.R, Ap.sup.S V. cholerae. The
final strain, V. cholerae JBK70, is sensitive to all antibiotics
tested, resistant to mercury, and phenotypically toxin negative.
Its chromosomal DNA did not detectably hydridize to DNA probes
containing CT genes. Short of sequencing the DNA for the entire
chromosome, JBK70 appears to be unaltered from the parent strain
N16961 except for the deletion of the toxin genes and insertion of
mercury resistance and inactive ampicillin resistance genes. Such a
strain cannot revert to toxigenicity because the toxin genes are
not merely mutated but are completely deleted.
EXAMPLE 5
Conjugal Gene Transfer to Confer Antitoxic Immunity
If both antibacterial immunity and antitoxic immunity are desired
for synergy, a derivative of JBK70 can be made to produce the B
subunit of cholera toxin only. To accomplish this end, a toxin
derivative was made that produces B only and lacks the genes for A
(FIG. 8). A Hpa II fragment from pJBK16 containing the B structural
gene was cloned into a phage cloning vector, M13mp7 placing a Bam
HI and an Eco RI site on either side of the gene (FIG. 8). The
fragment, now flanked by Bam HI sites was cloned into pMS 9 which
contains the very strong trp promoter. The placing of the B genes
under the transcriptional control of a strong promoter insures high
production of B antigen. Of the clones examined, approximately 50%
produced no antigen. This finding reflects the two possible
orientations for the cloned insert--one forward, one backward. One
derivative, pJBK51, which produced B subunit was mated into Vibrio
cholerae JBK70 and found to produce even more B antigen than the
parent strain N16961, yielding JBK70 (pJBK 51). Other B-only
mutants have been created using different promoters, including the
P.sub.L promoter and these can be evaluated in appropriate models
for any significant in vivo expression differences.
EXAMPLE 6
Colonization of Infant Mouse Intestine with JBK70 without Reversion
to Toxigenicity
Suckling mice (2.0-3.5g.) were removed from their mothers and
starved for 3 to 8 hours. Four of them were then inoculated on day
1 per os to stomach using a 22g animal feeding needle. The inoculum
was about 10.sup.8 CFU (colony-forming units)/mouse of JBK70 in a
volume of between 0.05 ml and 0.1 ml. The inoculum was prepared in
BHI broth essentially as described in Baselski, V. et al, supra.
The inoculum contained about 0.01% Evans blue dye. The presence of
this dye in the stomach, seen through the abdominal wall, indicated
proper delivery of the inoculum. Addition of Evans blue dye was
discontinued after day 1 (see Table I), to avoid inhibition of
JBK70.
Subsequent inoculations involved mouse-to-mouse (MXM), or
alternatively, mouse-to-plate-to-mouse (MXPXM), but required
different procedures to prepare the inoculum compared to the
Baselski protocol for the inoculation on day 1.
To prepare MXM inoculum, the gut was dissected from stomach to anus
under sterile precautions. The gut was weighed, placed in a glass
homogenizer tube, and about 0.5 ml BHI broth added. The mixture was
homogenized briefly with a Teflon pestle until tissue was
liquified. The resulting suspension was used to inoculate about
10.sup.-8 CFU into each infant mouse. It was checked for purity by
streaking on MEA (meat extract agar) plates. No Evans blue dye was
added.
To prepare MXPXM inoculum, a sterile loop was used to transfer
cells from an MEA plate to BHI broth. About 10.sup.11 CFU/ml were
added to about 1 ml of BHI so that a dense suspension was formed.
The mixture was vortexed to homogeneity, and 0.05-0.1 m. (about
10.sup.10 CFU) inoculated per os into each infant mouse. No evans
blue dye was added.
For all inoculations, mice were held in beakers at room temperature
of 73.degree.-76.degree. F. Beakers were placed in a plastic box
which was loosely covered in order to maintain the mice at slightly
above ambient temperature, about 78.degree. F.
As the results in Table I indicated, there were sufficient cells in
the intestine to inoculate the next animal, as checked by streaking
on MEA plates. The Vibrio cholerae JBK70 therefore colonized the
gut of infant mice. Furthermore, the fluid accumulation levels did
not increase since there were no substantial increases in the FA
ration (an FA ratio greater than or equal to 0.065 is a positive
fluid accumulation). Evidence of reversion to toxigenicity would
have indicated otherwise.
EXAMPLE 7
Construction of V. cholerae strain CVD101 having a Restriction
Fragment Deletion within the Gene coding for the A Subunit
Another classical strain chosen for attenuation was Vibrio cholerae
Ogawa 395 (alternatively designated "395") which, like N16961, has
been extensively studied in volunteers and confers solid immunity
[Levine, M. M. "Immunity to cholera as evaluated in volunteers," in
Cholera and Related Diarrheas: 43rd Nobel Symposium, Stockholm
1978. (O. Ouchterlong & J. Holmgren, eds.) Basel: S. Karger,
pp. 195-2-3 (1980); Levine, M. M et al. Acute Enteric, supra
(1981)]. The procedure employed in the attenuation of 395 was not
substantially different from that employed for N16961 (as described
in Examples 1-5).
The first step involved the cloning and mapping of the two toxin
gene copies of 395. Southern blot analysis revealed two Hind III
fragments of about 16 and about 12 kb in length, both of which
hybridized with cloned cholera toxin genes. These fragments were
purified by agarose gel electrophoresis and cloned into alkaline
phosphatase treated-Hind III digested pBR325 (FIG. 9). The
resulting recombinant plasmids containing the toxin genes were
designated pCVD14 and pCVD15.
Plasmids pCVD14 and pCVD15 were then mapped with restriction
endonucleases. An Xba I-Cla I fragment of about 550 bp was found,
containing the entire base sequence of the A.sub.1 subunit with the
exception of codons for the first 10 amino acid residues of
A.sub.1. This Xba I-Cla I fragment was deleted in vitro from both
pCVD14 and pCVD15 in a series of steps as shown in FIG. 10 for
pCVD15. First, partial digestion with Cla I yielded a population of
linear molecules in which only one of five Cla I sites was cut.
Next, the ends of the linear molecules were made blunt-ended by
filling in with DNA polymerase. Xba I linkers were ligated onto the
blunt-ended Cla I sites yielding a collection of molecules in which
an Xba I enzyme was then added to trim the linker and a
tetracycline resistance gene on an Xba I fragment was added and
ligated. After transformation into E. coli K-12 and selection on
tetracycline, the plasmid content of a number of transformants was
examined. A variety of deletion mutations were found in which one
or more Xba I-Cla I fragments were deleted. One deletion mutant was
chosen which lacked only the 550 bp Xba I-Cla I fragment containing
the A.sub.1 gene. This deletion mutant, designated pCVD25 was
purified, digested with Xba I and religated to delete the
tetracycline resistance gene. The resulting clone, PCVD30, was
negative for holotoxin as measured in Y-1 adrenal assay [Sack, D.
A. et. al. supra (1975)], but positive for production of B subunit,
as measured by ELISA [Sack, D. A. et al. supra (1980)], and lacked
the genes for A.sub.1, as shown by DNA hydridization using labeled
A.sub.1 probe. The Hind III fragment of pCVD30 containing the toxin
deletion mutation was then cloned into pJBK85, a Tc sensitive, Cm
resistant derivative of pJBK108. The resulting plasmid was
designated pJBK108.
The lack of a selectable marker in the toxin deletion mutation in
pJBK108 necessitated a modification of the method previously used
to attenuate El Tor N16961. To accomplish the deletion of the
A.sub.1 genes from 395, the Hind III fragment from pCVD15 was
cloned into pJBK85, resulting in pJBK88 (FIG. 11). The tetracycline
resistance gene on a Xba I fragment was then cloned into the Xba
site within the A.sub.1 gene of PJBK88, yielding pJBK107. This
tetracycline resistance was then recombined into the chromosome of
395 as previously done for V. cholerae pJBK56. pJBK107 (Tc.sup.r,
Cm.sup.r) was mobilized into 395 and a second Inc P plasmid, pR751
(Tp.sup.r) was introduced. Selection of Tc.sup.r, Tp.sup.r,
Cm.sup.s colonies resulted in V. cholerae JBK113, which contained
tetracycline resistance genes in both chromosomal toxin gene
copies. pJBK108, containing the deletion mutation, was then
mobilized into V. cholerae JBK113 . Homologous recombination of the
deletion mutation into the chromosome will result in the loss of
the A.sub.1 gene sequences, an event which can be detected by loss
of tetracycline resistance. Because the recombination event occurs
at a very low frequency, an enrichment procedure for tetracycline
sensitive cells in a population of tetracycline resistant cells was
employed. This enrichment procedure exploited the fact that
tetracycline is a bacteriostatic antibiotic whereas ampicillin and
D-cycloserine are bactericidal. Therefore, a culture of V. cholerae
JBK 113 containing pJBK108 was grown for 3 hr at 37.degree. in
L-broth containing 2 micro g/ml tetracycline, 50 micro g/ml
ampicillin and 50 micro g/ml D-cycloserine. At the end of 3 hours,
most of the tetracycline resistant cells were killed, and
tetracycline sensitive cells were detected by plating onto L-agar
and replica plating onto L-agar with tetracycline. Tetracycline
sensitive colonies were probed for the presence of A.sub.1 genes by
DNA hybridization. One tetracycline sensitive strain having
deletions for both gene copies of the A.sub.1 subunit was
designated V. cholerae CVD101 and tested for production of B
subunit by ELISA [Sack, supra]. V. cholerae CVD101 was found to
produce B subunit antigen at levels substantially equivalent to the
toxigenic parent V. cholerae 395.
EXAMPLE 8
DNA Sequencing of the Toxin Genes
The entire DNA sequence of the toxin genes of V. cholerae Inaba
62746 has been determined, part of which has been reported in
Lockman et al., J. Biol. Chem. 258, 13722 (1983). The restriction
endonuclease mapping of pCVD14 and pCVD15 indicates that the
sequences found in strain 62746 are also present in the toxin genes
of 395. The predicted junction after deletion of the 550 bp Xba
I-Cla I fragment, but with addition of an Xba I linker sequence, is
shown in FIG. 12. The Xba I site of the cholerae toxin sequence
spans amino acid residues 10 and 11 of the A.sub.1 structural gene
(not counting the 18 amino acid leader sequence for A.sub.1). The
Cla I site of the sequence is located at the last residue of
A.sub.1 and the first residue of A.sub.2.
EXAMPLE 9
Construction of V. Cholera CVD103
Another classical strain chosen for attenuation was V. cholerae
Inaba 569B (alternatively designated "569B"). The procedure
employed in the attenuation of 569B was not substantially different
from that employed for 395. The procedure used to prepare CVD103 is
the same as to prepare CVD101, except that 569B was used rather
than 395.
A 550 bp Xba I-Cla restriction fragment encoding 94% of the A.sub.1
peptide was deleted in vitro from the cloned cholera toxin genes.
This mutation was then recombined into the chromosome of 569B and
replaced the active toxin gene sequence of this strain. (As in
CVD101, two cholera toxin gene copies were replaced in the parent
strain. Vaccine strain V. cholerae JBK70 had only a single cholera
toxin gene copy).
The resulting strain, CVD103, has demonstrated its ability to
confer protective immunity with a minimum of reactogenicity. It is
clinically well tolerated, yet immunogenic and protective. Levine
et al., Lancet, pgs. 467-470 (August 1988), which is incorporated
herein by reference, reports challenge studies after vaccination by
CVD103 and reports vaccine efficacy of 87%, 82% and 67% for
different challenge strains at page 467, Table II, and lines 8 to
21. CVD103 caused only mild diarrhea in 6 of 52 volunteers and
these vaccinees did not suffer from the syndrome of abdominal
cramps, malaise, headache and nausea that had been seen in
approximately one-half of the recipients of other vaccine strains,
including JBK70, CVD101, and strain 395N1 disclosed in U.S. Pat.
No. 4,882,278 (Mekalanos). Vibrio cholerae strain CVD103Hg.sup.r
has been deposited with the American Type Culture Collection and
been given ATCC designation number 55456.
EXAMPLE 10
Construction of CVD103Hg.sup.r
CVD103Hg.sup.r is a derivative of CVD103. This strain contains a
mercury resistance marker. The mercury resistance marker allows the
strain to be distinguished from other strains of Vibrio cholerae
which could conceivably co-infect a recipient of the vaccine in a
cholera-endemic area such as Bangladesh or India. By inoculating a
plate of agar medium containing mercuric chloride, CVD103Hg.sup.r
is distinguished from other strains by its ability to grow on
mercury versus the growth inhibition seen with other strains on
this medium. To construct CVD103Hg.sup.r, the genes encoding the
hemolysin of V. cholerae (the hly genes) were first cloned into E.
coli . The hly sequences were digested with the restriction enzyme
HpaI and a 4.2 kilobase fragment encoding mercury resistance
originally derived from the plasmid R100 was cloned into the HpaI
site. Mutation of the hly gene at the HpaI site does not interfere
with the colonization and immunizing ability of V. cholerae.
(Kaper, et al., Advances in Research on Cholera and Related
Diarrheas, vol. 6, Y. Takeda and R. B. Sack, editors, KTK
Scientific Publishers, Tokyo, 1988, pps. 161-167.) The
construction, i.e., the cloned hly genes with the inserted mercury
resistance gene, was then mobilized into V. cholerae CVD103. Once
inside the bacterial cell, the mutated hly sequences recombined
with the homologous sequences in the CVD103 chromosome, and
resulted in the incorporation of the mercury resistance gene in the
chromosome of CVD103. The resulting strain was designated
CVD103Hg.sup.r.
CVD103Hg.sup.r has demonstrated its ability to provide significant
protection in challenge studies, and is even better tolerated than
CVD103 (Levine et al., The Lancet, "Safety, Immunogenicity, And
Efficacy Of Recombinant Live Oral Cholera Vaccines, CVD 103 and
CVD103Hgr", Aug. 27, 1988, pps 467-470.) CVD103Hg.sup.r has been
well-tolerated in randomized, double-blind, placebo-controlled
studies carried out in Swiss adults. Cryz et al., Vaccine 8, pgs.
577-579 (December 1990). Besides being well-tolerated,
CVD103Hg.sup.r exhibits a high degree of protective efficacy. The
best immunologic correlate of protection is seroconversion of
vibriocidal antibody. In North American studies, 95% of subjects
who received CVD103Hg.sup.r vaccine mounted a .gtoreq. fourfold
rise in Inaba vibrocidal antibody titre (Levine et al., supra), and
a response rate of 88% was seen in Swiss subjects (Cryz et al.,
Vaccine 8, at page 579). Thus, the high rate of seroconversion and
the good titres of both Inaba and Ogawa vibriocidal antibody
achieved after administration of a single dose of CVD103Hg.sup.r
suggest that a high level of protection may be conferred by
immunization with the dosage tested. Indeed, studies involving
volunteers immunized with a single dose of CVD103Hg.sup.r show that
they were significantly protected against experimental challenge
with classical biotype V. cholerae of either Inaba or Ogawa
serotype and against El Tor Inaba.
Experimental studies that have been carried out to assess the
protective efficacy of CVD103Hg.sup.r have used the following
pathogenic strains as challenge organisms: classical Inaba 569B; El
Tor Inaba N16961; El Tor Ogawa E7946. CVD103Hg.sup.r has provided
significant protection against diarrhea due to all of these
challenge strains of different biotypes and serotypes. Moreover,
protection was 100% against severe cholera (diarrheal purge of
.gtoreq.5.0 liters) and against moderate cholera (diarrheal purge
of .gtoreq.3.0 liters). Overall protection against any diarrhea
(.gtoreq.2.0 liters of loose stools) was 100% against the classical
challenge strain and 60% against El Tor challenge strains.
Applicants have carried out extensive clinical studies with
CVD103Hg.sup.r in healthy adults in the U.S.A., in Switzerland, and
in Thailand and in children in Indonesia.
While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of
further modification and this application is intended to cover any
variations, uses, or adaptations of the invention following, in
general, the principles of the invention and including such
departures from the present disclosure as come within known or
customary practice within the art to which the invention pertains
and as may be applied to the essential features hereinbefore set
forth, and as follows in the scope of the appended claims.
* * * * *